The vascular system plays a fundamentally important role in development, homeostasis and disease. With examples of scheduled vascular regression (the hyaloid vessels) and in the mouse, postnatal angiogenesis (in the retina), the eye is an excellent structure in which to study vascular development. In this application, we propose to study to role of retinal microglia, a type of myeloid cell, in the regulation of retinal angiogenesis. Based on published and preliminary studies, we propose the hypothesis that Retinal microglia regulate vascular pattern by producing VEGF and, via Wnts and possibly Notch, the VEGF inhibitor Flt1. We propose three aims to investigate this hypothesis: (1) to determine whether microglial or Muller glial VEGF mediates attractive guidance for deep layer retinal angiogenesis. Published work shows that Muller glia makes VEGF and our Preliminary Studies show that microglia also do. These are two possible sources of VEGF that might act as an attractive guidance cues for descending angiogenic sprouts. (2) To determine whether the non-canonical Wnt-Flt1 response of microglia is the Wnt-cGMP/Ca2+ pathway. Our Preliminary Studies suggest that the non-canonical Wnt-cGMP/Ca2+ pathway are involved in the production of Flt1 by microglia. We will further assess these using pharmacological inhibitors in a culture assay and by assessing the in vivo consequences of genetic deletion of pathway components. (3) To determine whether a microglial Notch response up-regulates Flt1 and integrates with the Wnt pathway. Published work and Preliminary Studies show that both VECs and microglia show Notch-dependent up-regulation of Flt1. This raises the possibility that when angiogenic tip cells (that express the Notch ligand Dll4 in the deep vascular layer), make contact with microglia, there is an up-regulation of Flt1. Given that Wnts also regulate Flt1 expression, it also suggests that the Wnt and Notch pathways must be integrated. These studies are important in uncovering basic mechanisms by which angiogenesis is normally regulated and as a result, uncovering new ways in which blood vessel growth and regression might be regulated therapeutically. In particular, this work has implications for the vision-compromising ailments diabetic retinopathy, age-related macular degeneration and retinopathy of prematurity. PUBLIC HEALTH RELEVANCE: In this application, we propose the study the role of macrophage-like cells called microglia in the development of blood vessels in the retina. We will study how microglia use growth factors called Wnt ligands to regulate the extent and pattern of blood vessels in the retina. This work has implications for the vascular diseases of the eye including diabetic retinopathy, the wet form of age-related macular degeneration and for retinopathy of prematurity.